Halogen-stabilized insulin

ABSTRACT

An insulin analogue comprises a B-chain polypeptide incorporating a halogenated phenylalanine at position B24, B25 or B26. The halogenated phenylalanine may be ortho-monofluoro-phenylalanine, ortho-monobromo-phenylalanine, ortho-monochloro-phenylalanine, or para-monochloro-phenylalanine. The analogue may be of a mammalian insulin, such as human insulin. A nucleic acid encodes such an insulin analogue. The halogenated insulin analogues retain significant activity. A method of treating a patient comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient. Halogen substitution-based stabilization of insulin may enhance the treatment of diabetes mellitus in regions of the developing world lacking refrigeration.

CROSS REFERENCED TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 13/018,011 filed Jan. 31, 2011, which is a continuation-in-partof International Application No. PCT/US2009/052477, filed Jul. 31, 2009,which claims benefit of U.S. Provisional Application No. 61/085,212filed on Jul. 31, 2008. Co-pending U.S. Ser. No. 13/018,011 is also acontinuation-in-part of International Application No. PCT/US2010/060085,filed Dec. 13, 2010, which claims benefit of U.S. ProvisionalApplication No. 61/285,955 filed on Dec. 11, 2009, which are allincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under cooperativeagreements awarded by the United States National Institutes of Healthunder grant numbers DK40949 and DK074176. The U.S. government may havecertain rights to the invention.

BACKGROUND OF THE INVENTION

This invention relates to polypeptides that are resistant to thermaldegradation. More particularly, this invention relates to thermallystabilized insulin analogues. Even more particularly, this inventionrelates to insulin analogues that are chemically and thermallystabilized by the incorporation of the element fluorine, chlorine, orbromine into an amino acid of the insulin analogue. These elements areclassified as halogens and are distinguished from the ordinaryconstituents of proteins by their atomic radius, electronegativity,stereoelectronic distribution of partial charges, transmitted effects onthe stereoelectronic properties of neighboring atoms in a molecule.

The engineering of ultra-stable proteins, including therapeutic agentsand vaccines, may have broad societal benefits in regions of thedeveloping world where electricity and refrigeration are notconsistently available. An example of a therapeutic protein susceptibleto thermal degradation is provided by insulin. The challenge posed byits chemical and physical degradation is deepened by the pendingepidemic of diabetes mellitus in Africa and Asia. Because chemicaldegradation rates of insulin analogs correlate inversely with theirrelative stabilities, the design of ultra-stable formulations mayenhance the safety and efficacy of insulin replacement therapy in suchregions.

The utility of some halogen substitutions in amino acids is wellestablished in medicinal chemistry. Indeed, fluorinated functionalgroups are critical to the efficacy of such widely prescribed smallmolecules as atorvastatin (Liptor™), an inhibitor of cholesterolbiosynthesis, and fluoxetine hydrochloride (Prozac™), a selectiveserotonin reuptake inhibitor used in the treatment of depression andother affective disorders. Although the atomic radius of fluorine issimilar to that of hydrogen, its large inductive effects modify thestereo-electronic properties of these drugs, in turn enhancing theirbiological activities. Such observations have motivated the study offluorinated amino acids in proteins. Similar considerations of physicalorganic chemistry pertain to the incorporation of larger halogen atoms,such as chlorine and bromine. The small molecule montelukast sodium(Singulair™) is a leukotriene inhibitor whose pharmaceutical propertiesare enhanced by covalent incorporation of a chlorine atom.

Attention has previously focused on the use of multiply fluorinatedaliphatic side chains (such as trifluoro-γ-CF₃-Val, trifluoro-δ-CF₃-Val,trifluoro-δ-CF₃-Ile, hexafluoro-γ_(1,2)-CF₃-Val, andhexafluoro-δ_(1,2)-CF₃-Leu) to maximize the gain in hydrophobicityassociated with this modification. An example is provided by thestabilization of a model α-helical motif, the homodimeric coiled coil.Its interfacial aliphatic chains were simultaneously substituted bytrifluoro-analogs, creating a fluorous core whose stability is enhancedby 0.3-2.0 kcal/mole. The degree of stabilization per fluorine atom is<0.1 kcal/mole. More marked stabilization per fluorine atom has beenachieved in an unrelated α-helical domain by substitution of a singleinternal Phe by pentafluoro-Phe (F₅-Phe)¹ (ΔΔG_(u) 0.6 kcal/mole perfive fluorine atoms). Stabilization occurs only at one specific positionin the protein, suggesting that its mechanism requires a particularspatial environment. The structure of the F₅-Phe-modified domain isidentical to that of the unmodified domain. Structural stabilizationhowever, is only a portion of the requirements for a biologically activepolypeptide. At least a significant portion of the activity must also bemaintained.

An extensive literature describes the use of fluorine labels in proteinsas ¹⁹F-NMR probe. Whereas such labels are widely regarded asnon-perturbing, applications of fluorinated amino acids in proteinengineering seek to exploit their altered physicochemical properties.Studies of a model α-helical fold (the villin headpiece subdomain)containing single F₅-Phe substitutions have demonstrated that whetherand to what extent such a modification may affect protein stabilitydepends in detail on structural environment. Indeed, the stability ofthis model fold was enhanced by an F₅-Phe substitution only at one ofthe seven sites tested in the core. It should be noted however, thatthis stabilizing effect was only demonstrated in a nonstandardpolypeptide analogue containing a sulfur atom in the main chain near thesite of fluorination; in our hands the self-same F₅-Phe substitution invillin headpiece subdomain with native polypeptide main chain has noeffect on its stability. Thus, to our knowledge, bona fide datademonstrating enhancement of protein stability due to halogenation of anaromatic residue in an otherwise native protein has not previously beenreported. These observations suggest that a generally hydrophobicenvironment does not in itself assure that the modification will bestabilizing. Because protein interiors are often stabilized byaromatic-aromatic interactions with a specific distance- and angulardependence, a subset of stabilizing F₅-Phe substitutions may adoptparticularly favorable perfluroaryl/aryl geometries. Such interactionsarise from the asymmetric distribution of partial charges in thesearomatic systems. Modulation of the chemical, physical, and biologicalproperties of proteins by the site-specific incorporation of chlorine orbromine atoms into modified amino acids are less well characterized inthe scientific literature than are the above effects of incorporation offluorine atoms.

Aromatic side chains may engage in a variety of weakly polarinteractions, involving not only neighboring aromatic rings but alsoother sources of positive- or negative electrostatic potential. Examplesinclude main-chain carbonyl- and amide groups in peptide bonds.

Administration of insulin has long been established as a treatment fordiabetes mellitus. Insulin is a small globular protein that plays acentral role in metabolism in vertebrates. Insulin contains two chains,an A chain, containing 21 residues and a B chain containing 30 residues.The hormone is stored in the pancreatic β-cell as a Zn²⁺-stabilizedhexamer, but functions as a Zn²⁺-free monomer in the bloodstream.Insulin is the product of a single-chain precursor, proinsulin, in whicha connecting region (35 residues) links the C-terminal residue of Bchain (residue B30) to the N-terminal residue of the A chain (FIG. 1A).Although the structure of proinsulin has not been determined, a varietyof evidence indicates that it consists of an insulin-like core anddisordered connecting peptide (FIG. 1B). Formation of three specificdisulfide bridges (A6-A11, A7-B7, and A20-B19; FIGS. 1A and 1B) isthought to be coupled to oxidative folding of proinsulin in the roughendoplasmic reticulum (ER). Proinsulin assembles to form solubleZn²⁺-coordinated hexamers shortly after export from ER to the Golgiapparatus. Endoproteolytic digestion and conversion to insulin occurs inimmature secretory granules followed by morphological condensation.Crystalline arrays of zinc insulin hexamers within mature storagegranules have been visualized by electron microscopy (EM).

Insulin functions in the bloodstream as a monomer, and yet it is themonomer that is believed to be most susceptible to fibrillation and mostforms of chemical degradation. The structure of an insulin monomer,characterized in solution by NMR, is shown in FIG. 1D. The A-chainconsists of an N-terminal α-helix (residues A1-A8), non-canonical turn(A9-A12), second α-helix (A12-A18), and C-terminal extension (A19-A21).The B chain contains an N-terminal arm (B1-B6), β-turn (B7-B10), centralα-helix (B9-B19), β-turn (B20-B23), β-strand (B24-B28), and flexibleC-terminal residues B29-B30. The two chains pack to form a compactglobular domain stabilized by three disulfide bridges (cystines A6-A11,A7-B7, and A20-B19).

Absorption of regular insulin is limited by the kinetic lifetime of theZn-insulin hexamer, whose disassembly to smaller dimers and monomers isrequired to enable transit through the endothelial lining ofcapillaries. The essential idea underlying the design of Humalog® andNovolog® is to accelerate disassembly. This is accomplished bydestabilization of the classical dimer-forming surface (the C-terminalanti-parallel β-sheet). Humalog® contains substitutions ProB28→Lys andLysB29→Pro, an inversion that mimics the sequence of IGF-I. Novolog®contains the substitution ProB28→Asp. Although the substitutions impairdimerization, the analogs are competent for assembly of a phenol- ormeta-cresol-stabilized zinc hexamer. This assembly protects the analogfrom fibrillation in the vial, but following subcutaneous injection, thehexamer rapidly dissociates as the phenol (or m-cresol) and zinc ionsdiffuse away. The instability of these analogs underlies their reducedshelf life on dilution by the patient or health-care provider. It wouldbe useful for an insulin analogue to augment the intrinsic stability ofthe insulin monomer while retaining the variant dimer-related β-sheet ofHumalog®.

Use of zinc insulin hexamers during storage is known and represents aclassical strategy to retard physical degradation and chemicaldegradation of a formulation in the vial or in the reservoir of a pump.Because the zinc insulin hexamer is too large for immediate passage intocapillaries, the rate of absorption of insulin after subcutaneousinjection is limited by the time required for dissociation of hexamersinto smaller dimers and monomer units. Therefore, it would advantageousfor an insulin analogue to be both (a) competent to permit hexamerassembly at high protein concentration (as in a vial or pump) and yet(b) sufficiently destabilized at the dimer interface to exhibitaccelerated disassembly—hence predicting ultra-rapid absorption from thesubcutaneous depot. These structural goals walk a fine line betweenstability (during storage) and instability (following injection).

Amino-acid substitutions in insulin have been investigated for effectson thermodynamic stability and biological activity. No consistentrelationship has been observed between stability and activity. Whereassome substitutions that enhance thermodynamic stability also enhancebinding to the insulin receptor, other substitutions that enhancestability impede such binding. The effects of substitution of Thr^(A8)by several other amino acids has been investigated in wild-type humaninsulin and in the context of an engineered insulin monomer containingthree unrelated substitutions in the B-chain (His^(B10)→Asp,Pro^(B28)→Lys, and Lys^(B29)→Pro) have been reported. Examples are alsoknown in the art of substitutions that accelerate or delay the timecourse of absorption. Such substitutions (such as Asp^(B28) in Novalog®and [Lys^(B28), Pro^(B29)] in Humalog®) can be and often are associatedwith more rapid fibrillation and poorer physical stability. Indeed, aseries of ten analogs of human insulin have been tested forsusceptibility to fibrillation, including Asp^(B28)-insulin andAsp^(B10)-insulin. All ten were found to be more susceptible tofibrillation at pH 7.4 and 37° C. than is human insulin. The tensubstitutions were located at diverse sites in the insulin molecule andare likely to be associated with a wide variation of changes inclassical thermodynamic stability. Although a range of effects has beenobserved, no correlation exists between activity and thermodynamicstability.

Insulin is a small globular protein that is highly amenable to chemicalsynthesis and semi-synthesis, which facilitates the incorporation ofnonstandard side chains. Insulin contains three phenylalanine residues(positions B1, B24, and B25). Conserved among vertebrate insulins andinsulin-like growth factors, the aromatic ring of Phe^(B24) packsagainst (but not within) the hydrophobic core to stabilize thesuper-secondary structure of the B-chain. Phe^(B24) lies at theclassical receptor-binding surface and has been proposed to direct achange in conformation on receptor binding.

The present theory of protein fibrillation posits that the mechanism offibrillation proceeds via a partially folded intermediate state, whichin turn aggregates to form an amyloidogenic nucleus. In this theory, itis possible that amino-acid substitutions that stabilize the nativestate may or may not stabilize the partially folded intermediate stateand may or may not increase (or decrease) the free-energy barrierbetween the native state and the intermediate state. Therefore, thecurrent theory indicates that the tendency of a given amino-acidsubstitution in the insulin molecule to increase or decrease the risk offibrillation is highly unpredictable.

Fibrillation, which is a serious concern in the manufacture, storage anduse of insulin and insulin analogues for diabetes treatment, is enhancedwith higher temperature, lower pH, agitation, or the presence of urea,guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drugregulations demand that insulin be discarded if fibrillation occurs at alevel of one percent or more. Because fibrillation is enhanced at highertemperatures, diabetic individuals optimally must keep insulinrefrigerated prior to use. Fibrillation of insulin or an insulinanalogue can be a particular concern for diabetic patients utilizing anexternal insulin pump, in which small amounts of insulin or insulinanalogue are injected into the patient's body at regular intervals. Insuch a usage, the insulin or insulin analogue is not kept refrigeratedwithin the pump apparatus and fibrillation of insulin can result inblockage of the catheter used to inject insulin or insulin analogue intothe body, potentially resulting in unpredictable blood glucose levelfluctuations or even dangerous hyperglycemia. At least one recent reporthas indicated that lispro insulin (an analogue in which residues B28 andB29 are interchanged relative to their positions in wild-type humaninsulin; trade name Humalog®) may be particularly susceptible tofibrillation and resulting obstruction of insulin pump catheters.

Insulin fibrillation is an even greater concern in implantable insulinpumps, where the insulin would be contained within the implant for 1-3months at high concentration and at physiological temperature (i.e. 37°C.), rather than at ambient temperature as with an external pump.Additionally, the agitation caused by normal movement would also tend toaccelerate fibrillation of insulin. In spite of the increased potentialfor insulin fibrillation, implantable insulin pumps are still thesubject of research efforts, due to the potential advantages of suchsystems. These advantages include intraperitoneal delivery of insulin tothe portal circulatory system, which mimics normal physiologicaldelivery of insulin more closely than subcutaneous injection, whichprovides insulin to the patient via the systemic circulatory system.Intraperitoneal delivery provides more rapid and consistent absorptionof insulin compared to subcutaneous injection, which can providevariable absorption and degradation from one injection site to another.Administration of insulin via an implantable pump also potentiallyprovides increased patient convenience. Whereas efforts to preventfibrillation, such as by addition of a surfactant to the reservoir, haveprovided some improvement, these improvements have heretofore beenconsidered insufficient to allow reliable usage of an implanted insulinpump in diabetic patients outside of strictly monitored clinical trials.

As noted above, the developing world faces a challenge regarding thesafe storage, delivery, and use of drugs and vaccines. This challengecomplicates the use of temperature-sensitive insulin formulations inregions of Africa and Asia lacking consistent access to electricity andrefrigeration, a challenge likely to be deepened by the pending epidemicof diabetes in the developing world. Insulin exhibits an increase indegradation rate of 10-fold or more for each 10° C. increment intemperature above 25° C., and guidelines call for storage attemperatures <30° C. and preferably with refrigeration. At highertemperatures insulin undergoes both chemical degradation (changes incovalent structure such as formation of iso-aspartic acid, rearrangementof disulfide bridges, and formation of covalent polymers) and physicaldegradation (non-native aggregation and fibrillation.

Amino-acid substitutions have been described in insulin that stabilizethe protein but augment its binding to the insulin receptor (IR) and itscross-binding to the homologous receptor for insulin-like growth factors(IGFR) in such a way as to confer a risk of carcinogenesis. An exampleknown in the art is provided by the substitution of His^(B10) byaspartic acid. Although Asp^(B10)-insulin exhibits favorablepharmaceutical properties with respect to stability andpharmacokinetics, its enhanced receptor-binding properties wereassociated with tumorigenesis in Sprague-Dawley rats. Although there aremany potential substitutions in the A- or B chains that can beintroduced into Asp^(B10)-insulin or related analogues to reduce itsbinding to IR and IGFR to levels similar to that of human insulin, suchsubstitutions generally impair the stability of insulin (or insulinanalogues) and increase its susceptibility to chemical and physicaldegradation. It would be desirable to discover a method of modificationof insulin and of insulin analogues that enabled “tuning” ofreceptor-binding affinities while at the same time enhancing stabilityand resistance to fibrillation. Such applications would require a set ofstabilizing modifications that reduce binding to IR and IGFR to varyingextent so as to offset the potential carcinogenicity of analogues thatare super-active in their receptor-binding properties.

Therefore, there is a need for alternative insulin analogues, includingthose that are stabilized during storage while maintaining at least aportion of the biological activity of the analogue.

SUMMARY OF THE INVENTION

It is, therefore, an aspect of the present invention to provide aninsulin analogue that provides greater stability by halogen substitutionin an amino acid, where the analogue than maintains at least a portionof biological activity of the corresponding non-halogenated insulin orinsulin analogue.

In addition or in the alternative, it is an aspect of the presentinvention to provide an insulin analogue that is a fast acting insulinbut also has improved stability over previous fast-acting insulinanalogues.

In general, the present invention provides an insulin analoguecomprising a B-chain polypeptide which incorporates a halogenatedphenylalanine at position B24, B25, or B26. In one embodiment, thehalogenated phenylalanine is located at position B24. In anotherembodiment, the halogenated phenylalanine at B24 is a chlorinatedphenylalanine or a fluorinated phenylalanine. In another embodiment, thehalogenated phenylalanine is ortho-monofluoro-phenylalanine,ortho-monobromo-phenylalanine, ortho-monochloro-phenylalanine orpara-monochloro-phenylalanine. In yet another embodiment, the insulinanalogue is a mammalian insulin analogue, such as an analogue of humaninsulin. In one particular set of embodiments, the B-chain polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ. ID. NOS. 4-8 and polypeptides having three or fewer additionalamino acid substitutions thereof.

Also provided is a nucleic acid encoding an insulin analogue comprisinga B-chain polypeptide which incorporates a halogenated phenylalanine atposition B24, B25, or B26. In one example, the halogenated phenylalanineis encoded by a stop codon, such as the nucleic acid sequence TAG. Anexpression vector may comprise such a nucleic acid and a host cell maycontain such an expression vector.

The invention also provides a method of treating a patient. The methodcomprises administering a physiologically effective amount of an insulinanalogue or a physiologically acceptable salt thereof to the patient,wherein the insulin analogue or a physiologically acceptable saltthereof contains a B-chain polypeptide incorporating a halogenatedphenylalanine at position B24, B25, or B26. In one embodiment, thehalogenated phenylalanine in the insulin analogue administered to apatient is located at position B24. In another embodiment, thehalogenated phenylalanine is ortho-monofluoro-phenylalanine,ortho-monobromo-phenylalanine, ortho-monochloro-phenylalanine, orpara-monochloro-phenylalanine. In still another embodiment, the insulinanalogue may be a mammalian insulin analogue, such as an analogue ofhuman insulin. In one particular set of embodiments, the B-chainpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ. ID. NOS. 4-8 and polypeptides having three or feweradditional amino acid substitutions thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a schematic representation of the sequence of humanproinsulin including the A- and B-chains and the connecting region shownwith flanking dibasic cleavage sites (filled circles) and C-peptide(open circles).

FIG. 1B is a structural model of proinsulin, consisting of aninsulin-like moiety and a disordered connecting peptide (dashed line).

FIG. 1C is a schematic representation of the sequence of human insulinindicating the position of residue B24 in the B-chain.

FIG. 1D is a ribbon model of an insulin monomer showing aromaticresidues in relation to the three disulfide bridges. The adjoining sidechains of Leu^(B15) (arrow) and Phe^(B24) are shown. The A- and B-chainchains are otherwise shown in light and dark gray, respectively, and thesulfur atoms of cysteines as circles.

FIG. 1E is a space-filling model of insulin showing the Phe^(B24) sidechain within a pocket at the edge of the hydrophobic core.

FIG. 2A is a representation of pentafluoro-phenylalanine (F₅-Phe).

FIG. 2B is a representation of ortho-monofluoro-phenylalanine (2F-Phe).

FIG. 2C is a representation of meta-monofluoro-phenylalanine (3F-Phe).

FIG. 2D is a representation of para-monofluoro-phenylalanine (4F-Phe).

FIG. 2E is a representation of ortho-monochlorinated-phenylalanine(2Cl-Phe).

FIG. 2F is a representation of meta-monochlorinated-phenylalanine(3Cl-Phe).

FIG. 2G is a representation of para-monochlorinated-phenylalanine(4Cl-Phe).

FIG. 3 is a set of four circular dichroism (CD) spectra for the far-UVwavelengths. Panel A: DKP-insulin (solid black line) and2F-Phe^(B24)-DKP (□); Panel B: DKP-insulin (solid black line) and3F-Phe^(B24)-DKP (▴); Panel C: DKP-insulin (solid black line) and4F-Phe^(B24)-DKP (▾); Panel D: DKP-insulin (solid black line) andF₅-Phe^(B24)-DKP ().

FIG. 4 is a set of four graphs for CD-detected guanidine denaturationstudies. Panel A: DKP-insulin (solid black line) and 2F-Phe^(B24)-DKP(□); Panel B: DKP-insulin (solid black line) and 3F-Phe^(B24)-DKP (▴);Panel C: DKP-insulin (solid black line) and 4F-Phe^(B24)-DKP (▾); PanelD: DKP-insulin (solid black line) and F₅-Phe^(B24)-DKP ().

FIG. 5 is a graph showing the results of receptor-binding studies ofinsulin analogs. Relative activities are determined by competitivebinding assay in which receptor-bound ¹²⁵I-labeled human insulin isdisplaced by increasing concentrations of DKP-insulin (▪) or itsanalogs: pentafluoro-Phe^(B24) (▴), 2F-Phe^(B24) (▾), 3F-Phe^(B24) (♦),and 4F-Phe^(B24) ().

FIG. 6 is a graph showing the results of receptor-binding studies ofinsulin analogs. Relative activities are determined by competitivebinding assay in which receptor-bound ¹²⁵I-labeled human insulin isdisplaced by increasing concentrations of KP-insulin (▪) or its analogs:2Br-Phe^(B24)-KP (▴), 2Cl-Phe^(B24)-KP (▾), 2F-Phe^(B24)-KP (♦), and4F-Phe^(B24)-KP (). Assay employed the B-isoform of the insulinreceptor and ¹²⁵I-Tyr^(A14)-human insulin as tracer.

FIG. 7A is a graph for CD-detected guanidine denaturation studies ofhuman insulin (solid line), KP-insulin (dashed line; “parent”), KPanalogues containing monofluorous substitution of 2F-Phe^(B24)-KP (▴),3F-Phe^(B24)-KP (▪), and 4F-Phe^(B24)-KP (◯).

FIG. 7B is a graph for CD-detected guanidine denaturation studies ofhuman insulin (solid line), KP-insulin (dashed line; “parent”),2-Cl-Phe^(B24)-KP (▾) and 2-Br-Phe^(B24)-KP (Δ).

FIG. 8A is a NMR spectra for DKP analogs comparing fluoro-substitutionsat positions 2, 3, and 4 of Phe^(B24) in relation to DKP-insulin,recorded at 700 MHz at 32° C. and pD 7.0.

FIG. 8B is a NMR spectra for KP analogs comparing fluoro-, bromo- andchloro-substitutions at position 2 of Phe^(B24) in relation toKP-insulin, recorded at 700 MHz at 32° C. and pD 7.0.

FIG. 9A is a graph showing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin(triangles), KP-insulin (squares), and 4Cl-Phe^(B24)-KP-insulin(inverted triangles).

FIG. 9B is a graph showing the results of in vitro receptor-bindingassays employing IGF-1R: human insulin (triangles), KP-insulin(squares), 4Cl-Phe^(B24)-KP-insulin (inverted triangles), and nativeIGF-I (circles).

FIG. 9C is a graph comparing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin (solidline), KP-insulin (dashed line), 4Cl-Phe^(B24)-KP-insulin (triangles)4F-Phe^(B24)-KP-insulin (squares).

FIG. 9D is a graph comparing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin (solidline), KP-insulin (dashed line), 4Cl-Phe^(B26)-KP-insulin (triangles)4F-Phe^(B26)-KP-insulin (squares).

FIG. 10 is a graph showing the hypoglycemic action of subcutaneous of4Cl-Phe^(B24)-KP-insulin in STZ induced diabetic Lewis rats over time(inverted triangles) relative to diluent alone (circles), human insulin(crosses), and KP-insulin (squares).

FIGS. 11A-C are graphs showing averaged traces of insulin cobaltsolutions showing characteristic spectral profiles from 400-750 nmbefore and after addition of 2 mM EDTA. Samples were dissolved in 50 mMTris (pH 7.4), 50 mM phenol, and 0.2 mM CoCl₂. NaSCN was then added to afinal concentration of 1 mM. Solid lines show data pre-EDTA extraction.Dashed lines show data post-EDTA extraction. Panel A: wild type insulin;Panel B: KP-insulin; Panel C; 4Cl-Phe^(B24)-KP-insulin.

FIG. 11D is a graph showing the kinetics of hexamer dissociation afteraddition of 2 mM EDTA as monitored at 574 nm (25° C. and pH 7.4). Datawere normalized to time zero for each sample: wild type (solid line),KP-insulin (dashed line), and 4Cl-Phe^(B24)-KP-insulin (dotted line).

FIG. 12 is a graph showing a plot of the mean filtered glucose infusionrate versus time after insulin dose for KP-insulin (Lispro insulin) and4Cl-Phe^(B24)-KP-insulin (4-Cl-Lispro insulin) at a dosage of 0.2 Unitsper kilogram of bodyweight.

FIG. 13 is a bar graph summarizing 20 pharmacodynamic studies in pigsdemonstrating significant improvement in ½ T-max late in4Cl-Phe^(B24)-KP-insulin over KP-insulin at five different dosinglevels.

FIG. 14 is a bar graph summarizing 14 pharmacodynamic studies in pigssuggesting improvement in ½ T-max early in 4Cl-Phe^(B24)-KP-insulin overKP-insulin at three different dosing levels.

FIG. 15 is a summary of ten, matched pharmacodynamics studies comparingthe relative potencies 4Cl-Phe^(B24)-KP-insulin with that of KP insulinas measured by area under the curve (AUC) in which the slightly reducedaverage potency for 4-Cl-KP was found not to be statisticallysignificant (p=0.22).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed an insulin analogue that providesgreater stability by halogen substitution in an amino acid, where theanalogue than maintains at least a portion of biological activity of thecorresponding non-halogenated insulin or insulin analogue. Particularly,the present invention provides insulin analogues that provides greaterstability by substitution of a single halogen in an amino acid, wherethe analogue than maintains at least a portion of biological activity ofthe corresponding non-halogenated insulin or insulin analogue. In oneexample, the present invention provides an insulin analogue thatprovides greater stability by fluorine, chlorine or bromine substitutionin an amino acid, where the analogue than maintains at least a portionof biological activity of the corresponding non-halogenated insulin orinsulin analogue. One potential application is to augment the chemicaland physical stability of an insulin analogue while retaining a portionof its biological activity; another application is to calibrate thereceptor-binding properties of an insulin analogue so not to exceed thatof human insulin. To these ends, the present invention provides insulinanalogues that contain a halogenated phenylalanine (Phe) residuesubstitution at position at B24, B25 or B26. While substitutions of ahalogenated phenylalanine at B24 or B25 do not change the basic aminoacid sequence of insulin or an insulin analogue, a halogenatedphenylalanine at position B26 of insulin must substitute for tyrosine(Tyr) found in the wild type sequence of insulin. In one particularembodiment, the present invention provides insulin analogues thatcontain a para-monochloro-phenylalanine (4Cl-Phe^(B24)) residue as asubstitution for wild type phenylalanine at position B24.

The present invention is not limited to human insulin and its analogueshowever. It is also envisioned that these substitutions may also be madein animal insulins such as porcine, bovine, equine, and canine insulins,by way of non-limiting examples.

Furthermore, in view of the similarity between human and animalinsulins, and use in the past of animal insulins in human diabeticpatients, it is also envisioned that other minor modifications in thesequence of insulin may be introduced, especially those substitutionsconsidered “conservative” substitutions. For example, additionalsubstitutions of amino acids may be made within groups of amino acidswith similar side chains, without departing from the present invention.These include the neutral hydrophobic amino acids: Alanine (Ala or A),Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline(Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) andMethionine (Met or M). Likewise, the neutral polar amino acids may besubstituted for each other within their group of Glycine (Gly or G),Serine (Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine(Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Basic aminoacids are considered to include Lysine (Lys or K), Arginine (Arg or R)and Histidine (His or H). Acidic amino acids are Aspartic acid (Asp orD) and Glutamic acid (Glu or E). Unless noted otherwise or whereverobvious from the context, the amino acids noted herein should beconsidered to be L-amino acids. In one example, the insulin analogue ofthe present invention contains three or fewer conservative substitutionsother than the halogenated-Phe substitutions of the present invention.In another example, the insulin analogue of the present inventioncontains one or fewer conservative substitutions other than thehalogenated-Phe substitutions of the present invention.

As used in this specification and the claims, various amino acids ininsulin or an insulin analogue may be noted by the amino acid residue inquestion, followed by the position of the amino acid, optionally insuperscript. The position of the amino acid in question includes the Aor B chain of insulin where the substitution is located. Thus, Phe^(B24)denotes a phenylalanine at the twenty fourth amino acid of the B chainof insulin, while Phe^(B25) denotes a phenylalanine at the twenty fifthamino acid of the B chain of insulin and Phe^(B26) denotes aphenylalanine substitution for tyrosine at the twenty sixth amino acidof the B chain of insulin. A fluorinated amino acid may be indicatedwith the prefix “F-,” a brominated amino acid may be indicated with theprefix “Br-” and a chlorinated amino acid may be indicated with theprefix “Cl-.” Therefore, fluorinated phenylalanine may be abbreviated“F-Phe,” chlorinated phenylalanine may be abbreviated “Cl-Phe” andbrominated phenylalanine may be abbreviated “Br-Phe.” In the case ofphenylalanine, the position of the fluorine substituents or substituentson the phenyl side chain may be further indicated by the number of thecarbon to which the fluorine is attached. Therefore,ortho-monofluoro-phenylalanine (shown in FIG. 2B) is abbreviated“2F-Phe,” meta-monofluoro-phenylalanine (shown in FIG. 2C) isabbreviated “3F-Phe” and para-monofluoro-phenylalanine (shown in FIG.2D) is abbreviated 4F-Phe. Pentafluoro-phenylalanine (shown in FIG. 2A)is abbreviated as “F₅-Phe.” Similarly, ortho-monobromo-phenylalanine maybe abbreviated “2Br-Phe,” ortho-monochloro-phenylalanine may (shown inFIG. 2E) be abbreviated “2Cl-Phe,” meta-monochloro-phenylalanine (shownin FIG. 2F) is abbreviated “3Cl-Phe” and para-monochloro-phenylalanine(shown in FIG. 2G) is abbreviated “4Cl-Phe.”

The phenylalanine at B24 is an invariant amino acid in functionalinsulin and contains an aromatic side chain. The biological importanceof Phe^(B24) in insulin is indicated by a clinical mutation (Ser^(B24))causing human diabetes mellitus. As illustrated in FIGS. 1D and 1E, andwhile not wishing to be bound by theory, Phe^(B24) is believed to packat the edge of a hydrophobic core at the classical receptor bindingsurface. The models are based on a crystallographic protomer (2-Znmolecule 1; Protein Databank identifier 4INS). Lying within theC-terminal β-strand of the B-chain (residues B24-B28), Phe^(B24) adjoinsthe central α-helix (residues B9-B19). One face and edge of the aromaticring sit within a shallow pocket defined by Leu^(B15) and Cys^(B19); theother face and edge are exposed to solvent (FIG. 1E). This pocket is inpart surrounded by main-chain carbonyl and amide groups and so creates acomplex and asymmetric electrostatic environment. The effects of thenear-symmetric substituent tetrafluoro-phenylalanine (F₅-Phe^(B24))(with its enhanced general hydrophobicity and fluoraryl-arylinteractions); (FIG. 2A) with those of “unbalanced” analogs containingsingle ortho-, meta-, or para-fluorous substitutions (designated 2F-Phe,3F-Phe, or 4F-Phe, FIGS. 2B-2D, respectively) on insulin stability areprovided.

Phe^(B25) is thought to lie at the surface of the insulin monomer and insolution structures projects into solvent with less structuralorganization than that of Phe^(B24). Tyr^(B26), like Phe^(B24), lies atthe edge of the hydrophobic core where it packs near nonpolar residuesIle^(A2), Val^(A3), and Val^(B12). Photo-cross-linking studies suggestthat the side chains of residues B24, B25, and B26 each contact theinsulin receptor. In dimers and hexamers of insulin residues, B24-B26are believed to participate in an intermolecular anti-parallel β-sheet.Aromatic side chains at these positions are thought to stabilize packingbetween monomers at this β-sheet interface.

It is envisioned that the substitutions of the present invention may bemade in any of a number of existing insulin analogues. For example, thehalogenated phenylalanine (H-Phe) substitutions provided herein may bemade in insulin analogues such as Lispro (KP) insulin, insulin Aspart,other modified insulins or insulin analogues, or within variouspharmaceutical formulations, such as regular insulin, NPH insulin, lenteinsulin or ultralente insulin, in addition to human insulin. Aspartinsulin contains an Asp^(B28) substitution and is sold as Novalog®whereas Lispro insulin contains Lys^(B28) and Pro^(B29) substitutionsand is known as and sold under the name Humalog®. These analogues aredescribed in U.S. Pat. Nos. 5,149,777 and 5,474,978, the disclosures ofwhich are hereby incorporated by reference herein. Both of theseanalogues are known as fast-acting insulins.

A halogenated-Phe substitution at B24, B25 and/or B26 may also beintroduced into analogues of human insulin that, while not previouslyclinically used, are still useful experimentally, such as DKP insulin,described more fully below, or miniproinsulin, a proinsulin analoguecontaining a dipeptide (Ala-Lys) linker between the A chain and B chainportions of insulin in place of the normal 35 amino acid connectingregion between the C-terminal residue of the B chain and the N-terminalresidue of the A chain (see FIG. 1B). Incorporation of halogenatedaromatic residues at positions B24, B25, or B26 in DKP-insulin (or otherinsulin analogues that contain Asp^(B10) or that exhibitreceptor-binding affinities higher than that of human insulin) canreduce their receptor-binding affinities to be similar to or below thatof human insulin and so potentially enable clinical use. In this mannerthe cross-binding of insulin analogues to the mitogenic IGFR may also bereduced.

The amino-acid sequence of human proinsulin is provided, for comparativepurposes, as SEQ. ID. NO. 1.

(proinsulin) SEQ. ID. NO. 1  Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln- Leu-Glu-Asn-Tyr-Cys-Asn

The amino acid sequence of the A chain of human insulin is provided asSEQ. ID. NO. 2.

(A chain) SEQ. ID. NO. 2  Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys- Asn

The amino acid sequence of the B chain of human insulin is provided asSEQ. ID. NO. 3.

(B chain) SEQ. ID. NO. 3 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

The amino acid sequence of a B chain of human insulin may be modifiedwith a substitution of a halogenated-Phe at position B24, B25, or B26.An example of such a sequence is provided As SEQ. ID. NO 4, where Xaamay be Tyr or Phe. The halogen used in any of these substitutions may befluorine, chlorine or bromine, for example.

SEQ. ID. NO. 4 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Xaa-Thr-Pro-Lys-Thr[Xaa is Tyr or Phe]

Further combinations of other substitutions are also within the scope ofthe present invention. It is also envisioned that the substitutions ofthe present invention may also be combined with substitutions of priorknown insulin analogues. For example, the amino acid sequence of ananalogue of the B chain of human insulin containing the Lys^(B28)Pro^(B29) substitutions of lispro insulin (Humalog®), in which one ofthe halogenated Phe substitution may also be introduced, is provided asSEQ. ID. NO. 5. Likewise, the amino acid sequence of an analogue of theB chain of human insulin containing the Asp^(B28) substitution of aspartinsulin, in which a F-Phe^(B24) or a Cl-Phe^(B24) substitution may alsobe introduced, is provided as SEQ. ID. NO. 6.

SEQ. ID. NO. 5 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Xaa-Thr-Lys-Pro-Thr[Xaa is Tyr or Phe] SEQ. ID. NO. 6Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Xaa-Thr-Asp-Lys-Thr[Xaa is Tyr or Phe]

The F-Phe^(B24) or Cl-Phe^(B24) substitution may also be introduced incombination with other insulin analogue substitutions such as analoguesof human insulin containing His substitutions at residues A4, A8 and/orB1 as described more fully in co-pending International Application No.PCT/US07/00320 and U.S. application Ser. No. 12/160,187, the disclosuresof which are incorporated by reference herein. For example, aF-Phe^(B24) or Cl-Phe^(B24) substitution may be present with [His^(A4),His^(A8)], and/or His^(B1) substitutions in an insulin analogue orproinsulin analogue having the amino acid sequence represented by SEQ.ID. NO. 7,

(SEQ. ID. NO. 7) R1-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-R2-Thr-R3-R4-Thr-Xaa⁰⁻³⁵-Gly-Ile-Val-R5-Gln-Cys-Cys-R6-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu- Asn-Tyr-Cys-Asn; 

wherein R1 is His or Phe; wherein R2 is Tyr or Phe, R3 is Pro, Lys, orAsp; wherein R4 is Lys or Pro; wherein R5 is His or Glu; wherein R6 isHis or Thr; and wherein Xaa₀₋₃₅ is 0-35 of any amino acid or a break inthe amino acid chain;

and further wherein at least one substitution selected from the group ofthe following amino acid substitutions is present:

-   -   R1 is His; and    -   R6 is His; and    -   R5 and R6 together are His.

A halogenated-Phe substitution at B24, B25, or B26 may also beintroduced into a single chain insulin analogue as disclosed inco-pending U.S. patent application Ser. No. 12/419,169, the disclosureof which is incorporated by reference herein.

It is also envisioned that the chlorinated-Phe^(B24) substitution may beintroduced into an insulin analogue containing substitutions in theinsulin A-chain. For example, an insulin analogue may additionallycontain a lysine, histidine or arginine substitution at position A8, asshown in SEQ. ID. NO. 27, instead of the wild type threonine at positionA8 (see SEQ. ID. NO. 2).

SEQ. ID. NO. 27 Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn [Xaa = His, Arg, or Lys]

Fluorine or chlorine substitutions such as those at B24 may beintroduced within an engineered insulin monomer of high activity,designated DKP-insulin, which contains the substitutions Asp^(B10) (D),Lys^(B28) (K) and Pro^(B29) (P). These three substitutions on thesurface of the B-chain are believed to impede formation of dimers andhexamers. Use of an engineered monomer circumvents confounding effectsof self-assembly on stability assays. The structure of DKP-insulinclosely resembles a crystallographic protomer. The sequence of theB-chain polypeptide for DKP insulin is provided as SEQ. ID. NO. 8, whereXaa is Tyr or Phe.

(DKP B-Chain Sequence) SEQ. ID. NO. 8 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-Asp-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Xaa-Thr-Lys-Pro-Thr

Analogues of DKP-insulin were prepared by trypsin-catalyzedsemi-synthesis and purified by high-performance liquid chromatography(Mirmira, R. G., and Tager, H. S., 1989. J. Biol. Chem. 264: 6349-6354.)This protocol employs (i) a synthetic octapeptide representing residues(N)-GF*FYTKPT (including modified residue (F*) and “KP” substitutions(underlined); SEQ. ID. NO. 9) and (ii) truncated analoguedes-octapeptide[B23-B30]-Asp^(B10) insulin (SEQ. ID. NO. 16). Becausethe octapeptide differs from the wild-type B23-B30 sequence (GFFYTPKT;SEQ. ID. NO. 10) by interchange of Pro^(B28) and Lys^(B29) (italics),protection of the lysine ε-amino group is not required during trypsintreatment. In brief, des-octapeptide (15 mg) and octapeptide (15 mg)were dissolved in a mixture of dimethylacetamide/1,4-butandiol/0.2 MTris acetate (pH 8) containing 10 mM calcium acetate and 1 mM ethylenediamine tetra-acetic acid (EDTA) (35:35:30, v/v, 0.4 mL). The final pHwas adjusted to 7.0 with 10 μL of N-methylmorpholine. The solution wascooled to 12° C., and 1.5 mg of TPCK-trypsin was added and incubated for2 days at 12° C. An additional 1.5 mg of trypsin was added after 24 hr.The reaction was acidified with 0.1% trifluoroacetic acid and purifiedby preparative reverse-phase HPLC (C4). Mass spectrometry usingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF;Applied Biosystems, Foster City, Calif.) in each case gave expectedvalues (not shown). The general protocol for solid-phase synthesis is asdescribed (Merrifield et al., 1982. Biochemistry 21: 5020-5031).9-fluoren-9-yl-methoxy-carbonyl (F-moc)-protected phenylalanine analogswere purchased from Chem-Impex International (Wood Dale, Ill.).

Circular dichroism (CD) spectra were obtained at 4° C. using an Avivspectropolarimeter (Weiss et al., Biochemistry 39: 15429-15440). Samplescontained ca. 25 μM DKP-insulin or analogs in 50 mM potassium phosphate(pH 7.4); samples were diluted to 5 μM for guanidine-induceddenaturation studies at 25° C. To extract free energies of unfolding,denaturation transitions were fitted by non-linear least squares to atwo-state model as described by Sosnick et al., Methods Enzymol. 317:393-409. In brief, CD data θ(x), where x indicates the concentration ofdenaturant, are fitted by a nonlinear least-squares program according to

${\theta (x)} = \frac{\theta_{A} + {\theta_{B}^{{({{{- \Delta}\; G_{H_{2}O}^{o}} - {mx}})}/{RT}}}}{1 + ^{{- {({{\Delta \; G_{H_{2}O}^{o}} - {mx}})}}/{RT}}}$

where x is the concentration of guanidine and where θ_(A) and θ_(B) arebaseline values in the native and unfolded states. Baselines areapproximated by pre- and post-transition lines θ_(A)(x)=θ_(A) ^(H) ²^(O)+m_(A)x and θ_(B)(x)=θ_(B) ^(H) ² ^(O)+m_(B)x.

Relative activity is defined as the ratio of analogue to wild-type humaninsulin required to displace 50 percent of specifically bound ¹²⁵I-humaninsulin. A human placental membrane preparation containing the insulinreceptor (IR) was employed as known in the art. Membrane fragments(0.025 mg protein/tube) were incubated with ¹²⁵I-labeled insulin (ca.30,000 cpm) in the presence of selected concentrations of unlabelledanalogue for 18 hours at 4° C. in a final volume of 0.25 ml of 0.05 MTris-HCl and 0.25 percent (w/v) bovine serum albumin at pH 8. Subsequentto incubation, mixtures are diluted with 1 ml of ice-cold buffer andcentrifuged (10,000 g) for 5 min at 4° C. The supernatant is thenremoved by aspiration, and the membrane pellet counted forradioactivity. Data is corrected for nonspecific binding (amount ofradioactivity remaining membrane associated in the presence of 1 μMhuman insulin. In all assays the percentage of tracer bound in theabsence of competing ligand was less than 15% to avoid ligand-depletionartifacts. An additional assay to monitor changes in activity during thecourse of incubation of the single-chain analogue at 37° C. wasperformed using a microtiter plate antibody capture as known in the art.Microtiter strip plates (Nunc Maxisorb) were incubated overnight at 4°C. with AU5 IgG (100 μl/well of 40 mg/ml in phosphate-buffered saline).Binding data were analyzed by a two-site sequential model. Acorresponding microtiter plate antibody assay using the IGF Type Ireceptor was employed to assess cross-binding to this homologousreceptor.

The far-ultraviolet circular dichroism (CD) spectra of the singlyfluorinated analogs are essentially identical to that of the parentanalogue (FIG. 3); a slight distortion is observed in the spectrum ofthe F₅-Phe^(B24) analogue (FIG. 3, Panel D). The stabilities andreceptor-binding activities of the analogs are provided in Table 1.Modified B24 residues were introduced within the context of DKP-insulin.Activity values shown are based on ratio of hormone-receptordissociation constants relative to human insulin; the activity of humaninsulin is thus 1.0 by definition. Standard errors in the activityvalues were in general less than 25%. Free energies of unfolding(ΔG_(u)) at 25° C. were estimated based on a two-state model asextrapolated to zero denaturant concentration. Lag time indicates time(in days) required for initiation of protein fibrillation on gentleagitation at 37° C. in zinc-free phosphate-buffered saline (pH 7.4).

TABLE 1 Activities and Stabilities of Insulin Analogs ΔG_(u) C_(mid) manalog activity (kcal/mole)^(c) (M) (kcal mol⁻¹ M⁻¹) lag timeDKP-insulin 2.42 4.0 ± 0.1 5.6 ± 0.1 0.70 ± 0.01 12.4 ± 2.5F₅-Phe^(B24)-DKP 0.013 4.8 ± 0.1 5.8 ± 0.1 0.84 ± 0.02 17.7 ± 2.12F-PheB²⁴-DKP 0.37 4.9 ± 0.1 5.8 ± 0.1 0.85 ± 0.02 16.0 ± 0.13F-Phe^(B24)-DKP 1.02 3.8 ± 0.1 5.5 ± 0.2 0.70 ± 0.02 17.7 ± 3.74F-Phe^(B24)-DKP 0.43 3.9 ± 0.1 5.6 ± 0.2 0.70 ± 0.02 11.0 ± 1.6

Substitution of Phe^(B24) by F₅-Phe augments the stability ofDKP-insulin by 0.8±0.1 kcal/mole at 25° C. (FIG. 4, Panel D). Despitesuch a favorable effect on stability, the activity of the analogue isnegligible (<1% receptor-binding affinity relative to the parent analog;Table 1 and FIG. 5). This impairment is more marked than that ordinarilyobserved on standard single-amino-acid substitution. By contrast, thesingly substituted analogs each retain significant activity: ortho (37%relative to human insulin), meta (100%), and para (43%). Such activitiesare each above the threshold of 10% (corresponding to a hormone-receptordissociation constant of <1 nM) generally predictive of therapeuticefficacy. Further, whereas the 3F-Phe^(B24) and 4F-Phe^(B24) analogs areno more stable (and possibly slightly less stable) than the parentanalogue (FIG. 4 and Table 1), 2F-Phe^(B24)-DKP-insulin is at least asstable as the F₅-Phe analogue (ΔΔG_(u) 0.9±0.1 kcal/mole). The physicalstabilities of the analogues were evaluated in triplicate duringincubation in 60 μM phosphate-buffered saline (PBS) at pH 7.4 at 37° C.under gentle agitation. The samples were observed for 20 days or untilsigns of precipitation or frosting of the glass vial were observed. F₅-,2F- and 3F-Phe^(B24)-DKP-insulin analogues (but not4F-Phe^(B24)-DKP-insulin) each exhibit an increase in lag time (relativeto the parent analogue) prior to onset of protein fibrillation (Table1). Substitution of a single fluorine atom is thus able to augment thestability of insulin with retention of reduced but clinicallysignificant activity; the modification can also extend the lag timeprior to protein fibrillation on gentle agitation at pH 7.4 and 37° C.

Whereas the F₅-Phe^(B24) substitution is highly stabilizing (similar tothe most favorable of the core substitutions in the villin subdomain),this insulin analogue is essentially without biological activity.Remarkably, the regiospecific modification 2F-Phe^(B24) is equallystabilizing while retaining substantial (although reduced) affinity forthe insulin receptor.

Fluorine substitutions were also introduced essentially as describedabove with the exception of ortho-, meta-, para-, and penta-fluorinatedphenylalanine being introduced at positions B24, B25 and B26 in lisproinsulin (that is, an insulin analogue also containing the substitutionsLys^(B28), Pro^(B29) (sold under the name Humalog®)). Analoguescontaining ortho-, meta-, para-, and penta-fluorinated phenylalaninesubstitutions at position B24 are designated 2F-Phe^(B24)-KP,3F-Phe^(B24)-KP, 4F-Phe^(B24)-KP, F₅-Phe^(B24)-KP, respectively.Analogues containing ortho-, meta-, para-, and penta-fluorinatedphenylalanine substitutions at position B25 are designated2F-Phe^(B25)-KP, 3F-Phe^(B25)-KP, 4F-Phe^(B25)-KP, F₅-Phe^(B25)-KP,respectively. Analogues containing ortho-, meta-, para-, andpenta-fluorinated phenylalanine substitutions at position B26(substituting for tyrosine) are designated 2F-Phe^(B26)-KP,3F-Phe^(B26)-KP, 4F-Phe^(B26)-KP, F₅-Phe^(B26)-KP, respectively.Additionally, ortho-bromo and ortho-chloro phenylalanine was substitutedinto lispro insulin at position B24. These analogues are designated2Br-Phe^(B24)-KP and 2Cl-Phe^(B24)-KP, respectively.

More specifically, for halogenated phenylalanine substitutions at B24, asynthetic octapeptide representing residues (N)-GF*FYTKPT (halogenatedphenylalanine residue indicated as “F*” and “KP” substitutions(underlined); SEQ ID NO. 9) and truncated analoguedes-octapeptide[B23-B30]-insulin (wild type at position B10, SEQ ID NO.17) were used. For fluorinated phenylalanine substitutions at B25, asynthetic octapeptide representing residues (N)-GFF*YTKPT (fluorinatedphenylalanine indicated again as “F*” and “KP” substitutions(underlined); SEQ ID NO. 9) and truncated analoguedes-octapeptide[B23-B30]-insulin (wild type at position B10; SEQ. ID. NO17) were used. For fluorinated phenylalanine substitutions at B26, asynthetic octapeptide representing residues (N)-GFFF*TKPT (fluorinatedphenylalanine indicated again as “F*” and “KP” substitutions(underlined); SEQ ID NO. 18) and truncated analoguedes-octapeptide[B23-B30]-insulin (wild type at position B10; SEQ. ID. NO17) were used. des-octapeptide (15 mg) and octapeptide (15 mg) weredissolved in a mixture of dimethylacetamide/1,4-butandiol/0.2 M Trisacetate (pH 8) containing 10 mM calcium acetate and 1 mM ethylenediamine tetra-acetic acid (EDTA) (35:35:30, v/v, 0.4 mL). The final pHwas adjusted to 7.0 with 10 μL of N-methylmorpholine. The solution wascooled to 12° C., and 1.5 mg of TPCK-trypsin was added and incubated for2 days at 12° C. An additional 1.5 mg of trypsin was added after 24 hr.The reaction was acidified with 0.1% trifluoroacetic acid and purifiedby preparative reverse-phase HPLC (C4). Mass spectrometry usingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF;Applied Biosystems, Foster City, Calif.) in each case gave expectedvalues. Relative activity and dissociation constants were determined asdescribed above.

The resulting data is presented in Table 2 for substitution of afluorinated phenylalanine at position B24 of insulin. For comparisonpurposes, wild type insulin and 2F-Phe^(B24)-DKP were tested under thesame conditions at 37° C. Data for testing at 37° C. are presented belowin Table 3. Data for substitutions of fluorinated phenylalanine atpositions B24, B25 and B26 and bromo- and chloro-substitutedphenylalanine in a lispro insulin analogue background are presentedbelow in Table 4.

TABLE 2 Stability and Activity of Phe^(B24)-DKP ΔG_(u) ΔΔG_(u) C_(mid) mreceptor Sample (Kcal/mol) (Kcal/mol) (M Gu-HCl) (Kcal/mol/M) bindingDKP-insulin  4.3 ± 0.06 / 5.4 ± 0.1 0.80 ± 0.01 161 2F-Phe^(B24)-DKP 4.9± 0.1 0.6 5.8 ± 0.1 0.85 ± 0.02 37 3F-Phe^(B24)-DKP 3.8 ± 0.1 −0.5 5.5 ±0.2 0.70 ± 0.02 102 4F-Phe^(B24)-DKP 3.9 ± 0.1 −0.4 5.6 ± 0.2 0.70 ±0.02 43 F5-Phe^(B24)-DKP 4.84 ± 0.1  0.54 5.8 ± 0.1 0.85 ± 0.02 1

TABLE 3 Stability and Activity of Insulin and Phe^(B24)-DKP [37° C.]ΔG_(u) ΔΔG_(u) C_(mid) m receptor Sample (Kcal/mol) (Kcal/mol) (MGu-HCl) (Kcal/mol/M) binding wt-insulin 2.4 ± 0.10 — 4.1 ± 0.17 0.57 ±0.06 100 2F-Phe^(B24)-DKP 3.1 ± 0.1  0.7 4.9 ± 0.1  0.64 ± 0.03

TABLE 4 Stability And Activity Of Halogenated-Phe Analogue Of LisproInsulin ΔG_(u) ΔΔG_(u) C_(mid) m receptor Sample (Kcal/mol) (Kcal/mol)(M Gu-HCl) (Kcal/mol/M) binding KP-insulin  3.0 ± 0.06 / 4.5 ± 0.1 0.61± 0.01 92 2F-Phe^(B24)-KP 3.8 ± 0.1 1.0 ± 0.16 4.9 ± 0.1 0.76 ± 0.02 173F-Phe^(B24)-KP 3.0 ± 0.1 0.2 ± 0.16  4.5 ± 0.15 0.67 ± 0.02 124F-Phe^(B24)-KP 2.75 ± 0.1  0.05 ± 0.16  4.4 ± 0.1 0.62 ± 0.02 32F₅-Phe^(B24)-KP 3.8 ± 0.1 1.0 ± 0.16 5.0 ± 0.1 0.76 ± 0.01 0.042F-Phe^(B25)-KP 3.3 ± 0.1 0.5 ± 0.16 4.3 ± 0.2 0.76 ± 0.03 153F-Phe^(B25)-KP 2.9 ± 0.1 0.1 ± 0.16 4.5 ± 0.2 0.65 ± 0.02 414F-Phe^(B25)-KP 2.9 ± 0.1 0.1 ± 0.16 4.5 ± 0.1 0.65 ± 0.01 78F₅-Phe^(B25)-KP 2.9 ± 0.1 0.1 ± 0.16 4.5 ± 0.2 0.65 ± 0.03 0.42F-Phe^(B26)-KP 1.7 ± 0.1 −1.1 ± 0.16  4.0 ± 0.2 0.43 ± 0.03 0.13F-Phe^(B26)-KP 3.3 ± 0.1 0.5 ± 0.16 4.86 ± 0.2  0.69 ± 0.03 174F-Phe^(B26)-KP 3.7 ± 0.1 0.9 ± 0.16 4.9 ± 0.2 0.75 ± 0.02 13F5-Phe^(B26)-KP 3.6 ± 0.2 0.8 ± 0.26  4.7 ± 0.26 0.76 ± 0.04 32Br-Phe^(B24)-KP  3.6 ± 0.05 0.8 ± 0.11 4.8 ± 0.1 0.75 ± 0.01 312Cl-Phe^(B24)-KP  3.8 ± 0.06 1.0 ± 0.12 4.9 ± 0.1 0.77 ± 0.01 36

Receptor-binding data for analogues of lispro insulin by competitivedisplacement are also presented in FIG. 6. The assay employed theB-isoform of the insulin receptor and ¹²⁵I-Tyr^(A14)-human insulin astracer.

Lispro insulin analogues containing either chloro- or bromo-substitutedphenylalanine at B24 (2Cl-Phe^(B24)-KP and 2Br-Phe^(B24)-KP,respectively) were tested for the ability to lower blood sugar indiabetic rats in comparison to non-halogenated lispro (KP) insulin. MaleLewis rats (˜250 g body weight) were rendered diabetic withstreptozotocin. Lispro insulin analogue, 2Cl-Phe^(B24)-KP and2Br-Phe^(B24)-KP were purified by HPLC, dried to powder, and dissolvedin insulin diluent (Eli Lilly Corp). Rats were injected subcutaneouslyat time=0 with 20 μg or 6.7 μg insulin analogue in 100 μl of diluent.Blood was obtained from clipped tip of the tail at time 0 and every 10minutes up to 90 min. Blood glucose was measured using a HypoguardAdvance Micro-Draw meter. Blood glucose concentration changes (inmilligrams (mg) per decaliter (dL) per hour (h)) are listed in Tables 5and 6 (these studies were performed with different sets of rats and soexhibit differences in baseline pharmacodynamic response to controlinjections of KP-insulin; top lines in each table).

TABLE 5 Blood Glucose Levels In Response To Halogenated Analogues OfLispro Insulin Insulin Analogue Δ Blood Glucose Sample (μg) (mg/dL/h)KP-insulin 20 191 +/− 20 KP-insulin 6.7 114 +/− 15 2Cl-Phe^(B24)-KP 20194 +/− 26 2Cl-Phe^(B24)-KP 6.7 179 +/− 30 2Br-Phe^(B24)-KP 20 224 +/−39 2Br-Phe^(B24)-KP 6.7 189 +/− 13

TABLE 6 Blood Glucose Levels In Response To Halogenated AnaloguesInsulin Analogue Δ Blood Glucose Sample (μg) (mg/dL/h) KP-insulin 6.7164 +/− 20 2F-Phe^(B24)-DKP 6.7 180 +/− 8  2F-Phe^(B24)-KP 6.7 131 +/−16 4F-Phe^(B26)-KP 6.7 164 +/− 17

As seen in Tables 5 and 6, the halogenated-phenylalanine containinginsulin analogues were equal or greater in potency than lispro(Humalog®) insulin with the exception of 2F-Phe^(B24)-KP-insulin. Thepotency of a 2F-Phe^(B24) insulin analog can be restored to that ofKP-insulin by inclusion of the Asp^(B10) substitution in the2F-Phe^(B24)-DKP-insulin analogue (line 2 of Table 6). Additionally, thepresence of a halogenated phenylalanine at position B24 increasesfibrillation lag time between three-fold and four-fold; thus, whereasKP-insulin under the above experimental conditions exhibits a lag timeof about 3 days, substitution of Phe^(B24) by either 2F-Phe^(B24),2Cl-Phe^(B24), or 2Br-Phe^(B24) extends the lag time to between 10-12days. These modifications also increase the thermodynamic stability (asprobed by guanidine denaturation) by between 0.5 and 1 kcal/mol (Table4).

Dissociation constants (K_(d)) were determined as described by Whittakerand Whittaker (2005. J. Biol. Chem. 280: 20932-20936), by a competitivedisplacement assay using ¹²⁵I-Tyr^(A14) insulin (kindly provided byNovo-Nordisk) and the purified and solubilized insulin receptor (isoformB) in a microtiter plate antibody capture assay with minor modification;transfected receptors were tagged at their C-terminus by a triple repeatof the FLAG epitope (DYKDDDDK; SEQ. ID. NO 11) and microtiter plateswere coated by anti-FLAG M2 monoclonal antibody (Sigma). The percentageof tracer bound in the absence of competing ligand was less than 15% toavoid ligand-depletion artifacts. Binding data were analyzed bynon-linear regression using a heterologous competition model (Wang,1995, FEBS Lett. 360: 111-114) to obtain dissociation constants.

TABLE 7 Cross-Binding of Insulin Analogues to the IGF Receptor proteinK_(d) (nM) relative to insulin IGF-I 0.037 ± 0.003 260 human insulin 9.6± 0.3 1 KP-insulin 10.6 ± 1   1 Asp^(B10)-insulin 4.2 2 Arg^(B31),Arg^(B32), Gly^(A21)-insulin 3.1 3 DKP-insulin 1.6 ± 0.1 62F-Phe^(B24)-KP 34 <0.3 2Br-Phe^(B24)-KP 10.1 1 2Cl-Phe^(B24)-KP 9.6 12F-Phe^(B24)-DKP 9.2 1

Measurements of cross-binding of selected insulin analogues to the IGFreceptor are summarized in Table 7. Human insulin under these conditionsbinds to IGFR 260-fold less tightly than does IGF-I. Cross-binding ofAsp^(B10)-insulin is increased by twofold whereas cross-binding by ananalogue in wide clinical use, Arg^(B31), Arg^(B32), Gly^(A21)-insulin(insulin glargine; Lantus™), is increased by threefold. Suchaugmentation of IGFR cross-binding is undesirable as treatment ofSprague-Dawley rats with Asp^(B10)-insulin is associated with anincreased incidence of mammary tumors whereas recent clinical studiessuggest the possibility that human use of Lantus confers adose-dependent increase in the risk of diverse cancers. (The sequence ofIGF-I contains Glu at position B10, similar in negative charge toAsp^(B10). While not wishing to be bound by theory, it is believed thatcross-binding by Lantus is augmented by the two basic residues at theC-terminus of the B-chain. Because IGF-I also contains Lys at positionB28 and Pro at position B29, it is possible that KP-insulin may exhibita small increase in cross-binding but the above data are notsufficiently precise to establish this.) DKP-insulin exhibits a six-foldincrease in IGFR cross-binding. Significantly, this increase iscounter-balanced by an ortho-monofluoro substitution at B24:2F-Phe^(B24)-DKP-insulin exhibits the same low level of cross-binding aswild-type human insulin. Its hypoglycemic potency in diabetic rats isalso the same as that of human insulin (Table 6). Because2F-Phe^(B24)-DKP-insulin is monomeric even at proteinconcentrations >0.5 mM, this analog is therefore of potential utility asan ultra-fast acting and ultra-stable insulin formulation for clinicaluse. Cross-binding affinities of 2Cl-Phe^(B24)-KP-insulin and2Br-Phe^(B24)-KP-insulin are also at a low level similar to that ofhuman insulin. These analogs are also fully active in diabetic rats(Table 5).

CD spectra were determined for human insulin, KP-insulin (lispro) andhalogenated Phe^(B24) insulin analogues as described above. CD-detectedprotein denaturation is provided as a function of the concentration ofguanidine hydrochloride in FIGS. 7A and 7B. FIG. 7A provides acomparison of human insulin (solid line) and KP-insulin (dashed line;“parent”) with KP analogues containing monofluorous substitution ofPheB24 at position 2, 3, or 4 (filled triangles, filled squares, andopen circles, respectively). FIG. 7B provides a comparison of humaninsulin (solid line) and KP-insulin (dashed line; “parent”) withanalogues containing 2-C1 or 2-Br modification of PheB24 (filledinverted triangles and open triangles, respectively). Inferredthermodynamic parameters are provided above in Table 4. Fractionalunfolding was monitored by mean residue ellipticity at 222 nm at 25° C.

NMR structures of selected analogs have been obtained to demonstratethat halogen substitutions are well accommodated in insulin and do notcause transmitted conformational perturbations. See FIGS. 8A and 8B. TheNMR structure of 2Cl-Phe^(B24)-KP-insulin as a monomer in solution isthus similar to that of KP-insulin; similarly, the NMR structure of2F-Phe^(B24)-DKP-insulin (also as a monomer in solution) is similar tothat of DKP-insulin. Qualitative analysis of NMR spectra of2F-Phe^(B24)-KP-insulin and 2Br-Phe^(B24)-KP-insulin likewise indicatenative-like structures.

Crystal structures have been determined of 2F-Phe^(B24)-KP-insulin and4F-Phe^(B24)-KP-insulin as zinc-stabilized hexamers in the presence ofthe preservative phenol (data not shown). The structures are similar tothose previously reported for KP-insulin as a zinc-stabilized hexamer inthe presence of phenol. In each case the conformation of the hexamer isT₃R^(f) ₃ containing two axial zinc ions and three bound phenolmolecules. Electron density for the halogen atoms is readily observed.The structure of the dimer- and trimer-forming surfaces are preserved inthe halogenated analogues, suggesting that pharmaceutical formulationsmay be obtained similar to those employed for KP-insulin and otherfast-acting insulin analogues.

Modified residues were introduced within the context of KP-insulin.Activity values shown are based on ratio of hormone-receptordissociation constants relative to human insulin; the activity of humaninsulin is thus 1.0 by definition. Standard errors in the activityvalues were in general less than 25%. Free energies of unfolding(ΔG_(u)) at 25° C. were estimated based on a two-state model asextrapolated to zero denaturant concentration. Lag time indicates time(in days) required for initiation of protein fibrillation on gentleagitation at 37° C. in zinc-free phosphate-buffered saline (pH 7.4).

The chlorinated-Phe^(B24) substitution provided herein may be made ininsulin analogues such as lispro insulin (that is, an insulin analoguealso containing the substitutions Lys^(B28), Pro^(B29) (sold under thename Humalog®)). Such an insulin analogue is designatedchlorinated-Phe^(B24)-KP-insulin. For comparative purposes, fluorinesubstitutions were also introduced, essentially as described above, withthe exception of para-fluorinated phenylalanine being introduced atpositions B24 and B26 in lispro insulin. Analogues containingpara-fluorinated phenylalanine substitutions at position B24 aredesignated 4F-Phe^(B24)-KP-insulin. Analogues containingpara-fluorinated phenylalanine substitutions at position B26(substituting for tyrosine) are designated 4F-Phe^(B26)-KP-insulin.Designations of respective analogues of KP-insulin and DKP-insulin maybe abbreviated in tables and figures as KP and DKP with “insulin”omitted for brevity.

For chlorinated phenylalanine substitutions at B24, a syntheticoctapeptide representing residues (N)-GF*FYTKPT (chlorinatedphenylalanine residue indicated as “F*” and “KP” substitutions(underlined); SEQ. ID. NO. 9) and truncated analoguedes-octapeptide[B23-B30]-insulin (wild type at position B10, SEQ. ID.NO. 17) were used. For fluorinated phenylalanine substitutions at B26, asynthetic octapeptide representing residues (N)-GFFF*TKPT (fluorinatedphenylalanine indicated again as “F*” and “KP” substitutions(underlined); SEQ. ID. NO. 18) and truncated analoguedes-octapeptide[B23-B30]-insulin (SEQ. ID. NO. 17) were used.

The resulting data for substitution of a halogenated phenylalanine atposition B24 or B26 in a lispro insulin analogue background arepresented below in Table 8.

TABLE 8 Stability And Activity Of Halogenated-Phe Analogues Of LisproInsulin ΔG_(u) ΔΔG_(u) C_(mid) m receptor Sample (Kcal/mol) (Kcal/mol)(M Gu-HCl) (Kcal/mol/M) binding (%) KP-insulin  3.0 ± 0.06 / 4.5 ± 0.10.61 ± 0.01 92 4F-Phe^(B24)-KP 2.75 ± 0.1  0.05 ± 0.16 4.4 ± 0.1 0.62 ±0.02 32 4F-Phe^(B26)-KP 3.7 ± 0.1  0.9 ± 0.16 4.9 ± 0.2 0.75 ± 0.02 134Cl-Phe^(B24)-KP 2.6 +/− 0.1 0.4 ± 0.2 4.7 +/− 0.2 0.58 +/− 0.02 90-100

Fibrillation Assays.

The physical stability of 4Cl-Phe^(B24)-KP-insulin was evaluated intriplicate during incubation in zinc-free phosphate-buffered saline(PBS) at pH 7.4 at 37° C. under gentle agitation in glass vials. Thesamples were observed for 12 days at a protein concentration of 60 μMfor visual appearance of cloudiness; twice daily aliquots were withdrawnfor analysis of thioflavin-T (ThT) fluorescence. Because ThTfluorescence is negligible in the absence of amyloid but is markedlyenhanced on onset of fibrillation, this assay probes for the lag time.Respective lag times for human insulin, KP-insulin, and4Cl-Phe^(B24)-KP-insulin are 51 days, 3±1 days, and more than 12 days.4Cl-Phe^(B24)-KP-insulin is therefore at least 4-fold more resistant tofibrillation under these conditions than is KP-insulin and at least2-fold more resistant than human insulin. While not wishing to conditionpatentability on theory, it is envisioned that increased fibrillationresistance of 4Cl-Phe^(B24) insulin analogues will allow them to beformulated in a zinc-free formulation to enhance the fast-acting natureof the insulin analogue without significantly shortening the storagetime of a sample of the analogue, either before or after an individualsample has begun to be used.

Thermodynamic Stability.

We measured the free energy of unfolding of 4Cl-Phe^(B24)-KP-insulinrelative to KP-insulin and LysA8-KP-insulin in a zinc-free buffer at pH7.4 and 25° C. (10 mM potassium phosphate and 50 mM KCl). This assayutilized CD detection of guanidine-induced denaturation as probed athelix-sensitive wavelength 222 nm. Values of ΔGu were extrapolated tozero denaturant concentration to obtain estimates by the free energy ofunfolding on the basis of a two-state model. Whereas the substitutionThrA8→Lys augmented thermodynamic stability by 0.6±0.2 kcal/mole, the4Cl-Phe^(B24) modification decreased stability by 0.4±0.2 kcal/mole.

The affinity of 4Cl-Phe^(B24)-KP-insulin-A8T for thedetergent-solubilized and lectin purified insulin receptor (isoform B)is similar to that of human insulin. A competitive displacement assayusing ¹²⁵I-labeled human insulin as tracer is shown in FIG. 9A usingisolated insulin receptor (isoform B): human insulin (triangles),KP-insulin (squares), 4Cl-Phe^(B24)-KP-insulin (inverted triangles). Allthree curves are closely aligned, indicating similar receptor-bindingaffinities. The affinity of 4Cl-Phe^(B24)-KP-insulin for the insulinreceptor was indistinguishable from that of KP-human insulin, in eachcase slightly lower than the affinity of wild-type insulin.

FIG. 9B shows results of corresponding assays employing Insulin-likeGrowth Factor I Receptor (IGF-1R), probed by competitive displacementusing ¹²⁵I-labeled IGF-I as tracer. Symbols are the same with theaddition of native IGF-I (circles). The rightward shift of the4Cl-Phe^(B24)-KP-insulin curve indicates decreased cross-binding toIGF-1R. The cross-binding of 4Cl-Phe^(B24)-KP-insulin to IGF-1R isreduced by approximately 3-fold relative to that of KP-insulin orwild-type insulin.

A similar comparison between human insulin (solid black line),KP-insulin (dashed line), 4Cl-Phe^(B24)-KP-insulin (triangles),4F-Phe^(B24)-KP-insulin (squares) using isolated insulin receptor(isoform B), is shown in FIG. 9C. The rightward shift of the4F-Phe^(B24)-KP-insulin curve relative to 4Cl-Phe^(B24)-KP-insulin, wildtype human insulin and lispro insulin shows decreased receptor-bindingaffinity with the use of a different halogen at the same position on thephenylalanine ring in comparison to a para-chloro substitution of thephenylalanine at B24. In contrast, the insulin receptor-binding affinityof 4Cl-Phe^(B24)-KP-insulin is similar to that of wild type humaninsulin and lispro (KP) insulin.

A further comparison between human insulin (solid line), KP-insulin(dashed line), 4Cl-Phe^(B26)-KP-insulin (triangles)4F-Phe^(B26)-KP-insulin (squares) using isolated insulin receptor(isoform B), is shown in FIG. 9D. Both 4Cl-Phe^(B26)-KP-insulin and4F-Phe^(B26)-KP-insulin show decreased insulin receptor-binding affinityin comparison to wild type and lispro insulins. As stated above,4Cl-Phe^(B24)-KP-insulin does not show a similar decrease inreceptor-binding affinity.

The in vivo potency of 4Cl-Phe^(B24)-KP-insulin in diabetic rats issimilar to that of KP-insulin. To enable characterization of biologicalactivity, male Lewis rats (˜300 g body weight) were rendered diabeticwith streptozotocin. Human insulin, KP-insulin, and4Cl-Phe^(B24)-KP-insulin were purified by HPLC, dried to powder, anddissolved in insulin diluent (Eli Lilly Corp). Rats were injectedsubcutaneously at time=0 with either 20 μg or 6.7 μg of KP-insulin or4Cl-Phe^(B24)-KP-insulin in 100 μl of diluent; the higher dose is at theplateau of the wild-type insulin dose-response curve whereas the lowerdose corresponds to 50-70% maximal initial rate of glucose disposal.Injection of diluent alone was performed as a negative control. 8 ratswere studies in each group. Blood was obtained from clipped tip of thetail at time 0 and at successive intervals up to 120 min. Blood glucosewas measured using a Hypoguard Advance Micro-Draw meter. Blood glucoseconcentrations were observed to decrease as shown in FIG. 10. Theinitial rate of fall of the blood glucose concentration during the first24 min after injection are similar on comparison of4Cl-Phe^(B24)-KP-insulin (−225±29 mg/dl/h), KP-insulin (−256±35mg/dl/h), and human insulin (−255±35 mg/dl/h). Any differences ininitial rate are not statistically significant. The duration of actionof 4Cl-Phe^(B24)-KP-insulin over the next 60 min appears shorter,however, than the durations of human insulin or KP-insulin.

Given the native receptor-binding affinity of 4Cl-Phe^(B24)-KP-insulin,it would be unusual for its potency to be less than that of humaninsulin. Indeed, insulin analogs with relative affinities in the range30-100% relative to wild-type typically exhibit native potencies invivo. It is formally possible, however, that the biological potency of4Cl-Phe^(B24)-KP-insulin is somewhat lower (on a molar basis) than thepotencies of human insulin or KP-insulin. If so, we note that any suchdecrease would be within the threefold range of the molar activities ofcurrent insulin products in clinical use (by convention respectiveinternational units (IU) are redefined to reflect extent of glucoselowering, leading to product-to-product differences in the number ofmilligrams or nanomoles per unit). It should be noted that the slowdecline in blood glucose concentration on control injection ofprotein-free diluent (brown dashed line in FIG. 10) reflects diurnalfasting of the animals following injection.

A surrogate marker for the pharmacokinetics of insulin hexamerdisassembly (designated the EDTA sequestration assay) employs cobaltions (Co²⁺) rather than zinc ions (Zn²⁺) to mediate hexamer assembly.Although Co²⁺ and Zn²⁺ hexamers are similar in structure, the cobalt ionprovides a convenient spectroscopic probe due to its unfilledd-electronic shell.

The principle of the assay is as follows. Solutions of R₆phenol-stabilized Co²⁺ insulin hexamers are blue due to tetrahedral Co²⁺coordination; on disassembly the protein solution is colorless asoctahedral Co²⁺ coordination by water or EDTA(ethylene-diamine-tetra-acetic acid; a strong chelator of metal ions)lacks optical transitions at visible wavelengths as a consequence ofligand field theory. The EDTA sequestration assay exploits thesespectroscopic features as follows. At time t=0 a molar excess of EDTA isadded to a solution of R₆ insulin hexamers or insulin analog hexamers.Although EDTA does not itself attack the hexamer to strip it of metalions, any Co²⁺ ions released in the course of transient hexamerdisassembly become trapped by the chelator and thus unavailable forreassembly. The rate of disappearance of the blue color (the tetrahedrald-d optical transition at 574 nm of the R-specific insulin-bound Co²⁺)thus provides an optical signature of the kinetics of hexamerdisassembly.

Averaged traces of insulin cobalt solutions showing characteristicspectral profiles from 400-750 nm were determined before and afteraddition of 2 mM EDTA (FIGS. 11A-C). Samples were dissolved in 50 mMTris (pH 7.4), 50 mM phenol, and 0.2 mM CoCl₂. NaSCN was then added to afinal concentration of 1 mM. The kinetics of hexamer dissociation afteraddition of 2 mM EDTA as monitored at 574 nm (25° C. and pH 7.4) arealso shown. The spectra of the analogues before EDTA extraction areshown as solid lines. Post-EDTA extraction, the spectra are displayed asdashed lines. Wild type is shown in Panel A, KP-insulin in Panel B, and4Cl-Phe^(B24)-KP-insulin as in Panel C.Data were normalized to time zerofor each sample.

On the one hand, the baseline optical absorption spectra of thehexameric cobalt complexes at t=0 are similar among wild-type insulinhexamers, KP insulin hexamers, and 4Cl-Phe^(B24)-KP-insulin hexamers(see FIGS. 11A-11C). The similar shapes and magnitudes of theserespective d-d electronic transitions imply that the metal ions are insimilar R₆-specific tetrahedral coordination sites in wild-type andvariant hexamers. This result is significant as it implies that4Cl-Phe^(B24)-KP-insulin remains competent for metal-ion-mediatedassembly and hence a zinc-based formulation.

The kinetics of hexamer dissociation after addition of 2 mM EDTA asmonitored at 574 nm (25° C. and pH 7.4) shows that the wild-type andvariant hexamers exhibit marked differences in rates of EDTA-mediatedCo²⁺ sequestration. As expected, the wild-type hexamer exhibits thegreatest kinetic stability (solid line in FIG. 11D), followed byKP-insulin (dashed-dotted line in FIG. 11D), and4-Cl-Phe^(B24)-KP-insulin (dotted line in FIG. 11D). Respectivehalf-lives are 481 sec (wild type), 363 sec (KP-insulin), and 66 sec(4Cl-Phe^(B24)-KP-insulin). The extent of acceleration induced by thepara-chloro-aromatic substitution is thus more profound than thatassociated with the “KP switch” of Lispro insulin (Humalog™). Becausediffusion of zinc ions from the site of subcutaneous injection isanalogous to the in vitro sequestration of cobalt ions in the EDTASequestration assay, these findings predict that4Cl-Phe^(B24)-KP-insulin will exhibit a marked acceleration ofabsorption.

The pharmacokinetic (PK) and pharacodynamic (PD) properties and potencyof 4-Cl-Phe^(B24)-KP-insulin were investigated in relation to wild-typeinsulin (Humulin™; Eli Lilly and Co.) and KP-insulin (Humalog™) inadolescent Yorkshire farm pigs (weight 25-45 kg). The wild type andKP-insulin were used as provided by the vendor (Eli Lilly and Co.) inU-100 strength. The 4-Cl-Phe^(B24)-KP-insulin was formulated in Lillydiluent with a ratio of protein to zinc ions similar to that of the wildtype and KP-insulin products; its strength was U-87. On the day ofstudy, each animal underwent anesthesia induction with Telazol and thengeneral anesthesia with isoflurane. Each animal was endotreacheallyintubated, and oxygen saturation and end-tidal expired CO₂ werecontinuously monitored. To block endogenous pancreatic α- and β-cellsecretion, pigs were given a subcutaneous injection of octreotideacetate (44 μg/kg) approximately 30 min before beginning the clamp studyand every 2 h thereafter. After IV catheters were placed and baselineeuglycemia was established with 10% dextrose infusion, an IV injectionof the insulin was given through the catheter. In order to quantifyperipheral insulin-mediated glucose uptake, a variable-rate glucoseinfusion was given to maintain a blood glucose concentration ofapproximately 85 mg/dl. Such a glucose infusion was typically requiredfor 5-8 h until the glucose infusion rate returned to the pre-insulinbaseline. Glucose concentrations were measured with a Hemocue 201portable glucose analyzer every 10 min (instrument error rate: 1.9%).The computerized protocol for glucose clamping was as described byMatthews et al. 2-ml blood samples for insulin assay was also obtainedaccording to the following schedule: from 0-40 min after insulindelivery: 5-minute intervals; from 50-140 min: 10-minute intervals, andfrom 160 min—to the point when GIR is back to baseline: 20-minintervals. For analysis of PK/PD, a 20-min moving mean curve fit andfilter was applied. PD was measured as time to early half-maximal effect(½ T_(max) Early), time to late half-maximal effect (½ T_(max) Late),time to maximal effect, and area-under-the-curve (AUC) over baseline.For each of these analyses, the fitted curve, not the raw data, wasused. Each of 3 pigs underwent 3 studies. The results of these studiesare provided in FIGS. 12-15.

4-Cl-Phe^(B24)-KP-insulin (abbreviated in FIG. 12 as 4-Cl-LisproInsulin) was found to exhibit a significantly less prolonged late “tail”than KP-insulin or wild-type insulin. The improved turn-off of insulinaction suggests a potential clinical benefit with regard to latepost-prandial hypoglycemia.

FIG. 13 summarizes 20 pharmacodynamic studies in pigs demonstratingsignificant improvement in ½ T-max late in 4Cl-Phe^(B24)-KP-insulin overKP-insulin at five different dosing levels, 0.05 U/kg, 0.1 U/kg, 0.2U/kg, 0.5 U/kg, and 1 U/kg.

FIG. 14 summarizes 14 pharmacodynamic studies in pigs suggestingimprovement in ½ T-max early in 4Cl-Phe^(B24)-KP-insulin over KP-insulinat three different dosing levels, 0.05 U/kg, 0.1 U/kg, 0.2 U/kg.

FIG. 15 summarizes ten, matched pharmacodynamics studies comparing therelative potencies 4Cl-Phe^(B24)-KP-insulin with that of KP insulin asmeasured by area under the curve (AUC) in which the slightly reducedaverage potency for 4-Cl-KP was found not to be statisticallysignificant (p=0.22). The pharmacokinetic (PK) and pharmacodynamic (PD)properties of 4-Cl-Phe^(B24)-KP-insulin in relation to wild-type insulinand KP-insulin (Lispro-insulin) under similar formulation conditions(zinc insulin hexamers or zinc insulin analog hexamers stabilized byphenol and meta-cresol) show that the potency of4-Cl-Phe^(B24)-KP-insulin, as measured by area-under-the-curve (AUC)method, was similar to those of wild-type insulin and KP-insulin.

A method for treating a patient comprises administering ahalogen-substituted insulin analogue to the patient. In one example, thehalogen-substituted insulin analogue is an insulin analogue containinghalogen-substituted phenylalanine, such as a fluoro-, chloro- orbromo-substituted phenylalanine. In one particular example the halogensubstituted phenylalanine is 2F-Phe^(B24), 2F-Phe^(B25), 2F-Phe^(B26) or4Cl-Phe^(B24). In another example, the halogen-substituted insulinanalogue additionally contains one or more substitutions elsewhere inthe insulin molecule designed to alter the rate of action of theanalogue in the body. The insulin analogue may optionally contain ahistidine, lysine or arginine substitution at position A8. In stillanother example, the insulin analogue is administered by an external orimplantable insulin pump. An insulin analogue of the present inventionmay also contain other modifications, such as a tether between theC-terminus of the B-chain and the N-terminus of the A-chain as describedmore fully in co-pending International Application No. PCT/US07/080467(U.S. patent application Ser. No. 12/419,169), the disclosure of whichis incorporated by reference herein.

A pharmaceutical composition may comprise such insulin analogues andwhich may optionally include zinc. Zinc ions may be included in such acomposition at a level of a molar ratio of between 2.2 and 3.0 perhexamer of the insulin analogue. In such a formulation, theconcentration of the insulin analogue would typically be between about0.1 and about 3 mM; concentrations up to 3 mM may be used in thereservoir of an insulin pump. Modifications of meal-time insulinanalogues may be formulated as described for (a) “regular” formulationsof Humulin™ (Eli Lilly and Co.), Humalog™ (Eli Lilly and Co.), Novalin™(Novo-Nordisk), and Novalog™ (Novo-Nordisk) and other rapid-actinginsulin formulations currently approved for human use, (b) “NPH”formulations of the above and other insulin analogues, and (c) mixturesof such formulations. As mentioned above, it is believed that theincreased resistance to fibrillation will permit4Cl-Phe^(B24)-containing insulin analogues to be formulated without thepresence of zinc to maximize the fast acting nature of the analogue.However, it is also believed that even in the presence of zinc, the4Cl-Phe^(B24)-containing insulin analogues will dissociate from hexamersinto dimers and monomers sufficiently quickly as to also be considered afast-acting insulin analogue formulation.

Excipients may include glycerol, glycine, other buffers and salts, andanti-microbial preservatives such as phenol and meta-cresol; the latterpreservatives are known to enhance the stability of the insulin hexamer.Such a pharmaceutical composition may be used to treat a patient havingdiabetes mellitus or other medical condition by administering aphysiologically effective amount of the composition to the patient.

A nucleic acid comprising a sequence that encodes a polypeptide encodingan insulin analogue containing a sequence encoding at least a B-chain ofinsulin with a fluorinated phenylalanine at position B24, B25 or B26 isalso envisioned. This can be accomplished through the introduction of astop codon (such as the amber codon, TAG) at position B24 in conjunctionwith a suppressor tRNA (an amber suppressor when an amber codon is used)and a corresponding tRNA synthetase, which incorporates a non-standardamino acid into a polypeptide in response to the stop codon, aspreviously described (Furter, 1998, Protein Sci. 7:419-426; Xie et al.,2005, Methods. 36: 227-238). The particular sequence may depend on thepreferred codon usage of a species in which the nucleic acid sequencewill be introduced. The nucleic acid may also encode other modificationsof wild-type insulin. The nucleic acid sequence may encode a modified A-or B-chain sequence containing an unrelated substitution or extensionelsewhere in the polypeptide or modified proinsulin analogues. Thenucleic acid may also be a portion of an expression vector, and thatvector may be inserted into a host cell such as a prokaryotic host celllike an E. coli cell line, or a eukaryotic cell line such as S.cereviciae or Pischia pastoris strain or cell line.

For example, it is envisioned that synthetic genes may be synthesized todirect the expression of a B-chain polypeptide in yeast Piscia pastorisand other microorganisms. The nucleotide sequence of a B-chainpolypeptide utilizing a stop codon at position B24 for the purpose ofincorporating a halogenated phenylalanine at that position may be eitherof the following or variants thereof:

(a) with Human Codon Preferences: (SEQ. ID. NO. 12)TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTAGTTCTACACACCCAAGACC(b) with Pichia Codon Preferences: (SEQ. ID. NO. 13)TTTGTTAACCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTAGTTTTACACTCCAAAGACT

Similarly, a full length pro-insulin cDNA having human codon preferencesand utilizing a stop codon at position B24 for the purpose ofincorporating a halogenated phenylalanine at that position may have thesequence of SEQ. ID NO. 14.

(SEQ. ID. NO. 14) TTTGTGAACC AACACCTGTG CGGCTCACAC CTGGTGGAAG CTCTCTACCT AGTGTGCGGG GAACGAGGCT AGTTCTACAC ACCCAAGACC CGCCGGGAGG CAGAGGACCT GCAGGTGGGGCAGGTGGAGC TGGGCGGCGG CCCTGGTGCA GGCAGCCTGCAGCCCTTGGC CCTGGAGGGG TCCCTGCAGA AGCGTGGCAT TGTGGAACAA TGCTGTACCA GCATCTGCTC CCTCTACCAG CTGGAGAACT ACTGCAACTA G

Likewise, a full length human pro-insulin cDNA utilizing a stop codon atposition B24 for the purpose of incorporating a halogenatedphenylalanine at that position and having codons preferred by P.pastoris may have the sequence of SEQ. ID. NO 15.

(SEQ. ID. NO. 15) TTTGTTAACC AACATTTGTG TGGTTCTCAT TTGGTTGAAGCTTTGTACTT GGTTTGTGGT GAAAGAGGTT AGTTTTACACTCCAAAGACT AGAAGAGAAG CTGAAGATTT GCAAGTTGGTCAAGTTGAAT TGGGTGGTGG TCCAGGTGCT GGTTCTTTGCAACCATTGGC TTTGGAAGGT TCTTTGCAAA AGAGAGGTAT TGTTGAACAA TGTTGTACTT CTATTTGTTC TTTGTACCAA TTGGAAAACT ACTGTAACTA A

Similarly, a nucleotide sequence of a B-chain polypeptide utilizing astop codon at position B25 for the purpose of incorporating ahalogenated phenylalanine at that position may be either of thefollowing or variants thereof:

(a) with Human Codon Preferences: (SEQ. ID. NO. 19)TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTAGTACACACCCAA GACC(b) with Pichia Codon Preferences: (SEQ. ID. NO. 20)TTTGTTAACCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTTTAGTACACTCCAAA GACT

A full length pro-insulin cDNA having human codon preferences andutilizing a stop codon at position B25 for the purpose of incorporatinga halogenated phenylalanine such as fluorinated phenylalanine at thatposition may have the sequence of SEQ. ID NO. 21.

(SEQ. ID. NO. 21) TTTGTGAACC AACACCTGTG CGGCTCACAC CTGGTGGAAGCTCTCTACCT AGTGTGCGGG GAACGAGGCT TCTAGTACACACCCAAGACC CGCCGGGAGG CAGAGGACCT GCAGGTGGGGCAGGTGGAGC TGGGCGGCGG CCCTGGTGCA GGCAGCCTGCAGCCCTTGGC CCTGGAGGGG TCCCTGCAGA AGCGTGGCATTGTGGAACAA TGCTGTACCA GCATCTGCTC CCTCTACCAG CTGGAGAACT ACTGCAACTA G

Likewise, a full length human pro-insulin cDNA utilizing a stop codon atposition B25 for the purpose of incorporating a halogenatedphenylalanine such as a fluorinated phenylalanine at that position andhaving codons preferred by P. pastoris may have the sequence of SEQ. ID.NO 22.

(SEQ. ID. NO. 22) TTTGTTAACC AACATTTGTG TGGTTCTCAT TTGGTTGAAGCTTTGTACTT GGTTTGTGGT GAAAGAGGTT TTTAGTACACTCCAAAGACT AGAAGAGAAG CTGAAGATTT GCAAGTTGGTCAAGTTGAAT TGGGTGGTGG TCCAGGTGCT GGTTCTTTGCAACCATTGGC TTTGGAAGGT TCTTTGCAAA AGAGAGGTATTGTTGAACAA TGTTGTACTT CTATTTGTTC TTTGTACCAA TTGGAAAACT ACTGTAACTA A

Likewise, a nucleotide sequence of a B-chain polypeptide utilizing astop codon at position B26 for the purpose of incorporating ahalogenated phenylalanine at that position may be either of thefollowing or variants thereof:

(a) with Human Codon Preferences: (SEQ. ID. NO. 23)TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTGCTCTACCTAGTGTGCGGGAACGAGGCTTCTTCTAGACACCC AAGACC(b) with Pichia Codon Preferences: (SEQ. ID. NO. 24)TTTGTTAACCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTTTTTTAGACTCCA AAGACT

A full length pro-insulin cDNA having human codon preferences andutilizing a stop codon at position B26 for the purpose of incorporatinga halogenated phenylalanine at that position may have the sequence ofSEQ. ID NO. 25.

(SEQ. ID. NO. 25) TTTGTGAACC AACACCTGTG CGGCTCACAC CTGGTGGAAGCTCTCTACCT AGTGTGCGGG GAACGAGGCT TCTTCTAGACACCCAAGACC CGCCGGGAGG CAGAGGACCT GCAGGTGGGGCAGGTGGAGC TGGGCGGCGG CCCTGGTGCA GGCAGCCTGCAGCCCTTGGC CCTGGAGGGG TCCCTGCAGA AGCGTGGCAT TGTGGAACAA TGCTGTACCA GCATCTGCTC CCTCTACCAG CTGGAGAACT ACTGCAACTA G

Likewise, a full length human pro-insulin cDNA utilizing a stop codon atposition B26 for the purpose of incorporating a halogenatedphenylalanine at that position and having codons preferred by P.pastoris may have the sequence of SEQ. ID. NO 26.

(SEQ. ID. NO. 26) TTTGTTAACC AACATTTGTG TGGTTCTCAT TTGGTTGAAGCTTTGTACTT GGTTTGTGGT GAAAGAGGTT TTTTTTAGACTCCAAAGACT AGAAGAGAAG CTGAAGATTT GCAAGTTGGTCAAGTTGAAT TGGGTGGTGG TCCAGGTGCT GGTTCTTTGCAACCATTGGC TTTGGAAGGT TCTTTGCAAA AGAGAGGTAT TGTTGAACAA TGTTGTACTT CTATTTGTTC TTTGTACCAA TTGGAAAACT ACTGTAACTA A

Other variants of these sequences, encoding the same polypeptidesequence, are possible, given the synonyms in the genetic code.

Based upon the foregoing disclosure, it should now be apparent thathalogen-substituted insulin analogues will carry out the objects setforth hereinabove. Namely, these insulin analogues exhibit enhancedthermodynamic stability, resistance to fibrillation and potency inreducing blood glucose levels. The halogen substitutedphenylalanine-containing insulin analogues also have reducedcross-reactivity to insulin-like growth factor (IGFR). It is, therefore,to be understood that any variations evident fall within the scope ofthe claimed invention and thus, the selection of specific componentelements can be determined without departing from the spirit of theinvention herein disclosed and described.

The following literature is cited to demonstrate that the testing andassay methods described herein would be understood by one of ordinaryskill in the art.

-   Furter, R., 1998. Expansion of the genetic code: Site-directed    p-fluoro-phenylalanine incorporation in Escherichia coli. Protein    Sci. 7:419-426.-   Merrifield, R. B., Vizioli, L. D., and Boman, H. G. 1982. Synthesis    of the antibacterial peptide cecropin A (1-33). Biochemistry 21:    5020-5031.-   Mirmira, R. G., and Tager, H. S. 1989. Role of the phenylalanine B24    side chain in directing insulin interaction with its receptor:    Importance of main chain conformation. J. Biol. Chem. 264:    6349-6354.-   Sosnick, T. R., Fang, X., and Shelton, V. M. 2000. Application of    circular dichroism to study RNA folding transitions. Methods    Enzymol. 317: 393-409.-   Wang, Z. X. 1995. An exact mathematical expression for describing    competitive biding of two different ligands to a protein molecule    FEBS Lett. 360: 111-114.-   Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and    Katsoyannis, P. G. 2000. Hierarchiacal protein “un-design”:    insulin's intrachain disulfide bridge tethers a recognition α-helix.    Biochemistry 39: 15429-15440.-   Whittaker, J., and Whittaker, L. 2005. Characterization of the    functional insulin binding epitopes of the full length insulin    receptor. J. Biol. Chem. 280: 20932-20936.-   Xie, J. and Schultz, P. G. 2005. An expanding genetic code. Methods.    36: 227-238.

1. An insulin analogue comprising a B-chain polypeptide incorporating amono-halogenated phenylalanine at position B24.
 2. The insulin analogueof claim 1, wherein the halogenated phenylalanine is anortho-mono-halogenated phenylalanine.
 3. The insulin analogue of claim2, wherein the analogue is an analogue of a mammalian insulin.
 4. Theinsulin analogue of claim 3, wherein the analogue is an analogue ofhuman insulin.
 5. A method of treating a patient comprisingadministering a physiologically effective amount of an insulin analogueor a physiologically acceptable salt thereof to the patient, wherein theinsulin analogue or a physiologically acceptable salt thereof contains aB-chain polypeptide incorporating a mono-halogenated phenylalanine atposition B24
 6. The method of claim 5, wherein the mono-halogenatedphenylalanine is an ortho-mono-halogenated phenylalanine.
 7. A nucleicacid encoding an insulin analogue according to claim
 3. 8. A nucleicacid encoding an insulin analogue according to claim 7, wherein thehalogenated phenylalanine is encoded by a stop codon.
 9. A nucleic acidencoding an insulin analogue according to claim wherein the nucleic acidis expressed in conjunction with a suppressor tRNA and a correspondingtRNA synthetase.
 10. The nucleic acid of claim 9, wherein the stop codonis the nucleic acid sequence TAG.
 11. An expression vector comprisingthe nucleic acid sequence of claim
 10. 12. A host cell transformed withthe expression vector of claim
 11. 13. A method of incorporating amonohalogenated phenylalanine at position B24 of a B-chain polypeptideof an insulin analogue, the method comprising expressing a nucleic acidencoding the insulin analogue, wherein the monohalogenated phenylalanineis encoded by a stop codon, wherein the nucleic acid is expressed inconjunction with a suppressor tRNA and a corresponding tRNA synthetasewhich incorporates the non-standard amino acid in response to the stopcodon.
 14. The method of claim 13, wherein the stop codon is the nucleicacid sequence TAG.
 15. The method of claim 14, wherein the nucleic acidsequence comprises any one of SEQ ID NOS: 12-15.
 16. The method ofclaims 15, wherein the nucleic acid is a portion of an expressionvector.
 17. The method of claim 16, wherein the expression vector isinserted into a host cell.
 18. The method of claims 14, wherein thenucleic acid is a portion of an expression vector.
 19. The method ofclaim 18, wherein the expression vector is inserted into a host cell.